Trivalent poliomyelitis live virus vaccine



United States atent TRIVALENT POLIOMYELITIS LIVE VIRUS VACCINE Herald Rea Cox, Suffern, N.Y., assignor to American Cyanamid Company, New York, N.Y., a corporation of Maine No Drawing. Filed June 22, 1959, Ser. No. 821,720

3 Claims. (Cl. 167--78) This invention relates to an improved trivalent attenuated live virus poliomyelitis vaccine suitable for oral administration.

The use of attenuated live virus polio vaccine is rapidly growing outside of the United States as well as inside and presents many advantages over the killed virus vaccine of the type pioneered by Dr. Salk. The safety of the attenuated live virus vaccine, when administered orally, has been completely proven by tests in South America on more than six-hundred thousand patients without any incidence of paralytic polio due to the vaccine. Excellent antibody response and a high degree of immunity has resulted. However, problems are presented. A large number of the vaccinations carried out in South America were effected by feeding orally separate strains of attenuated vaccine at short intervals. While this requires only three oral feedings and therefore presents real advantage over the necessary three or four injections with killed virus vaccine, it is still not ideal because it triples the time required for vaccination and enhances the possibility of patients forgetting to come back for one or two of the later vaccinations.

It was early realized that if a trivalent vaccine could be developed, that is to say a vaccine which was a mixture of attenuated strains of all three types of polio virus, this would be ideal. Unfortunately, attempts to develop a practically useable trivalent vaccine encountered serious problems along two lines which may be called interference phenomena and effectiveness, although to some extent the phenomena have certain common causes.

The interference phenomena arose in their most serious form when Dr. Sabin attempted to prepare and to use a trivalent vaccine using the three attenuated strains of Types I, II and III polio virus which he had developed. He found very serious interference because his attenuated Type II strain appeared to be dominant and inhibited or reduced very seriously the development of infections by Type I and TypeIII polio and hence the production of antibodies for these three types. This was particularly tragic because Type II is of minor importance compared to Types I and III, Type I being the great menace because of its frequent and predominant association with paralytic polio, Type III is of second importance and Type II of only minor importance, causing a very small percentage of paralytic outbreaks. Sabins trivalent vaccine was therefore useless practically and in his description of his experiments in the Journal of the American Medical As sociation, volume 162, pages 1589 to 1596, he concludes with the following statement: Immunization against all three types of polio virus by a single administration of a mixture of all three, is not feasible. He therefore recommended that the three strains be fed separately at three week intervals in the order Type I, Type III and finally Type II.

The problem of interference of attenuated Type II virus with Types I and III is completely solved in a triple vaccine using as its Type II attenuated strain, the strain developed in the Lederle Laboratories of the American Cyanamid Company by passage through the nervous tissue 2,966,443 Patented Dec. 27, 1960 of suckling rodents until adapted to grow in chick embryo and then successive passages through chick embryo. This is described in articles by Roca-Garcia et al., Poliomyelitis II: Propagation of MEF Strain of Poliomyelitis Virus in Developing Chick Embryo by Yolk Sac Inoculation, Proc. Soc. Exp. Biol. & Med. 81: 519, 1952; Cabasso et al.; Poliomyelitis II: Propagation of MEF Strain of Poliomyelitis Virus in Developing Chick Embryo by Allantoic Cavity Inoculation, Proc. Soc. Exp. Biol. & Med. 81: 525, 1952; and Cabasso et al., Poliomyelitis IV: Some Culture and Other Characteristics of Chick Embryo Adapted Type II Strain of Poliomyelitis Virus, Proc. Soc. Exp. Biol. & Med. 85: 167-171, 1954. This attenuated strain is also described and claimed in the copending application of Cabasso et al., Serial No. 783,007, filed December 26, 1958.

Triple vaccines prepared from the above referred to chick embryo adapted attenuated Type II strain and attenuated Type I virus of the SM strain and Type III virus of the Fox strain developed at the Lederle Laboratories above referred to and also described, for example, on pages 134 to 136 of the University of Minnesota Medical Bulletin, volume 29, December 1957, showed none of the problem of dominance of Type II, which rendered 'the Sabin experiments unsuccessful, but it presented another problem. When fed in a concentration of less than a million TCD of each type of virus, the percentage of individuals in the sero negative adult group developing antibodies was disappointing. Vaccine concentrations are customarily referred to by the common logarithm of the infective doses and this terminology will be used throughout the specification and claims. Experiments on a number of adults with triple vaccine containing each of three types of virus at log 5.8 showed antibody development in sero negatives at the rate of 71% for Type I, 67% for Type II and 75% for Type III. The above results were with adults in whom it is more diflicult to elicit antibody response with live virus vaccine. While this does not compare unfavorably with results with killed virus vaccines even when three shots are administered, it still leaves a distressingly large proportion of sero negatives unprotected, particularly against Types I and III which are the really dangerous ones.

Further work resulted in the surprising discovery that there is a very sharp threshold at log 6. At log 6.1, the percentages in adults were 96% for Type I and 100% for Type III. Type II increased to 78% and can be increased by still larger doses. The above test includes the routine vaccination in Minnesota hospitals of pregnant women who are notoriously more susceptible to paralytic polio. Thus, for the first time, there is available a triple vaccine giving almost 100% protection to sero negatives for the dangerous Type I and Type III strains and very substantial protection for the relatively much less serious Type II.

The present invention therefore depends, in its broader aspects, on two factors, (1) the use of a suckling rodent nervous tissue modified Type II strain vaccine which. shows no dominance and (2) doses not less than log 6. Why the suckling rodent modified Type II virus, and particularly the one which shows adaptation to growth in chick embryo, does not dominate whereas Dr. Sabins modified Type II strain does is, of course, not known. Viruses cannot be analyzed chemically to give any meaningful result andcan only be characterized by their biological properties and by the history of their development. Therefore, the present invention is not intended to be limited to any theory of why the Type II modified virus of the present invention is not dominant. Similarly, the existence of the sharp threshold at log 6 concentration is entirely unexpected and applicant has no knowledge as to why such a threshold exists for the trivalent vaccine of the present invention with the particular type of modified strain Type II, which does not dominate. Both factors are necessary because Dr. Sabins unsuccessful triple vaccine was fed at log 6 concentration and it was not effective. However, it had a dominant Type 11 strain and so, of course, did not meet one of the two requirements of the present invention.

The invention will be described in greater detail in conjunction with the following specific examples.

EXAMPLE 1 Preparation of the attenuated virus Type I, Sickles-Mahony strain, derived from a mixture of the Sickles strain obtained from Dr. Albert Sabin and the Mahony strain from Dr. Brown at the University of Michigan, was passed 26 successive times through mouse spinal tissue. This was followed by successive passages through monkey kidney tissue culture at which time the virus was adapted to grow in chick embryo. The virus was passed 14 successive times through chick embryo tissues, after which it was alternated in passage several times through monkey kidney tissue and chick embryo tissue. This constituted the Type I, avirulent seed, which was then grown in monkey kidney tissue to produce large quantities of material for blending in the final vaccine.

The Type II virus was obtained by taking MEF strain (originally isolated in the Middle East in 1942). It was passed nine times intracerebrally through mice and then 157 times through the nervous tissue of suckling hamsters. After these passages, the virus became adapted to growth in chick embryo and was then serially passed 17 times through chick embryo and 11 times through monkey kidney tissue. It was then plaqued in monkey kidney cells, passed twice, and this repeated. The resulting avirulent virus was seed virus for producing Type II vaccine by cultivation in monkey kidney tissue.

Type III virus was obtained by taking the Fox strain of avirulent virus. This was merely cultivated with occasional intermediate plaquing in monkey kidney cells and constituted the seed for Type III vaccine, which was then grown in monkey kidney tissue.

The tissue culture fluid of each virus harvest was diluted with an equal volume of 70% aqueous sorbitol solution. The fluid was then diluted with 35% aqueous sorbitol solution; the final syrup base diluent had the following composition and was prepared as indicated below:

Syrup base 35% sorbitol, ingredients per liter:

Methylparabens, U.S.P gms 0.44 Propylparabens, U.S.P. gms 0.11 Sodium phosphate, monobasic gms 2.76 Sodium phosphate, dibasic gms 11.36 Givandan Cherry flavor gms 0.26 Distilled water ml 500.00 Sorbitol solution 70%, q.s ml 500.00

Procedure for preparing the sorbitol solution was as follows: Heat distilled water to 9095 C., add parabens with stirring and dissolve, add the phosphates and dissolve, add the cherry flavoring, cool to 25 C., adjust volume q.s. with sorbitol solution 70%, filter through clarifying filter, check and adjust pH to 7.0:02, fill into sterile containers, assign log number and date. The final product was obtained and represents a trivalent vaccine having a titer of log 5.8 per cc. of each virus type.

One cc. of the above mixture was fed to a series of volunteers, mostly adult, the age percentage being as follows: 8 to 10 years, 7%; 11 through 20, 17%; 21 through 30, 22%; 31 through 40, 33%; 41 through 50,

14%; 51 through 60, 5%; and 61 through 65, 2%. Two percent of the volunteers were seronegative to all three types, 10% were negative to two types and 21% to a single type. These are referred to as triple negatives, double negatives and single negatives, respectively. After feeding, 71% of Type I seronegatives, 67% of Type II seronegatives and 75% of Type III seronegatives developed adequate antibodies.

Antibodies were determined by the method of Salk et al., Am. J. Hyg., 60 (2): pages 214-240, September 1954, and the results were calculated by the standard method of Reed and Muench, Am. J. Hyg., vol. 23, pages 493-497, 1938. Accordingly, where there was no virus neutralization by the serum on a dilution of 1:4 the person was referred to as a seronegative.

EXAMPLE 2 The procedure of Example 1 was repeated but in this case the final vaccine dose was 2 cc., in other words, log 6.1 of each virus type. The number of volunteers tested was approximately four times as great as in Example 1 and the age distribution to the nearest whole percent was as follows: 4 through 10, 2%; 11 through 20, 5%; '21 through 30, 33%; 31 through 40, 44%; 41 through 50, 10%; and 51 through 60, 5%. About 5% were triple negatives, 18% double negatives and single negatives, 26%. After vaccination, 96% of the Type I seronegatives, 78% of the Type II seronegatives and 100% of the Type III seronegatives developed antibodies.

The examples show vaccines prepared in the form of aqueous sorbitol solutions. This is a simple, reliable and palatable form but it should be understood that the invention is in no sense limited thereto and the triple vaccine may be incorporated by adsorption into unhydrated gelatin or mixed with other palatable diluents in solid or liquid form. Of course, the diluents not only have to be harmless to humans but must not be toxic to the attenuated viruses of the vaccine.

I claim:

1. A trivalent live poliomyelitis vaccine suitable for oral administration comprising a mixture of Type I virus attenuated to avirulence to man by successive passages through the nervous tissue of rodents, Type II virus adapted to chick embryo propagation by successive passages through the nervous tissue of suckling rodents and then attenuated through chick embryos until avirulent to man and Type III virus avirulent to man, the trivalent vaccine being in dosage unit form containing at least log 6 of each virus, the Type II virus of the mixture not inhibiting infection by the other two.

2. A trivalent vaccine according to claim 1 in which the viruses of the three types are dispersed in an aqueous sorbitol solution.

3. A process of preparing a trivalent live poliomyelitis vaccine which comprises attenuating Type I poliomyelitis virus to avirulence to man by successive passages through nervous tissue of rodents and attenuating Type II poliomyelitis virus by successive passage through the nervous tissue of suckling rodents until it is adapted for growth in chick embryo and then passing through chick embryos until avirulence to man, and mixing the two viruses with a Type III poliomyelitis virus avirulent to man to produce a dose containing the three types each in a concentration of at least log 6.

References Cited in the file of this patent Dick: British Med. J., Nov. 15, 1958, pp. 1184-1188. 

1. A TRIVALENT LIVE POLIOMYELITIS VACCINE SUITABLE FOR ORAL ADMINISTRATION COMPRISING A MIXTURE OF TYPE I VIRUS ATTENUATED TO AVIRULENCE TO MAN BY SUCCESSIVE PASSAGES THROUGH THE NERVOUS TISSUE OF RODENTS, TYPE II VIRUS ADAPTED TO CHICK EMBRYO PROPAGATION BY SUCCESSIVE PASSAGES THROUGH THE NERVOUS TISSUE OF SUCKLING RODENTS AND THEN ATTENUATED THROUGH CHICK EMBRYOS UNTIL AVIRULENT TO MAN AND TYPE III VIRUS AVIRULENT TO MAN, THE TRIVALENT VACCINE BEING IN DOSAGE UNIT FORM CONTAINING AT LEAST LOG 6 OF EACH VIRUS, THE TYPE II VIRUS OF THE MIXTURE NOT INHIBITING INFECTION BY THE OTHER TWO. 